The Program Project grant (yr 11-15) Signaling of Endothelial Permeability and Lung Vascular Injury addresses the critical mechanisms that mediate the loss of pulmonary vascular barrier function, in turn, inducing pulmonary edema. We have approached this subject in a multidisciplinary manner bringing to bear technologies of molecular and cellular biology, biochemistry, and cell imaging and functional analysis of the regulation of lung vascular barrier function in the mouse model. Project 1 addresses the potentially important and novel relationship between endothelial caveolin-1, the structural and signaling protein of caveolae, and adherens junctions (AJs), the structures known to regulate permeability of the junctions based on the postulate of a cross-talk between caveolae and AJs via eNOS. Project 2 addresses the mechanisms of a newly identified post-translational activation of iNOS activity and how the inordinately high-output NO generated regulates lung endothelial barrier function. Project 3 focuses on the signaling mechanisms by which TRPC6 (a prototypic receptor-operated calcium channel) activates both RhoA and the endothelial MLCK isoform to increase lung endothelial permeability. Project 4 addresses the fundamental observation of increased binding affinity on endothelial cell plasmalemma of the adhesive protein ICAM-1 and the feed-forward signaling mechanism of ICAM-1 activation of caveolae-mediated transcytosis and lung endothelial hyper-permeability. Our overall goal on the basis of these four highly interactive projects is to define signaling pathways regulating the increase in lung vascular permeability so that rational targets are identified for therapeutic purposes. Moreover the depth of understanding to be gained of the signaling pathways mediating the increase in lung vascular permeability will provide novel insights into the mechanisms of protein-rich pulmonary edema and ARDS.